The sheepshead minnow, Cyprinodon variegatus, is a widespread fish species that typically inhabits coastal tidal marsh and mangrove swamp environments, ranging from Cape Cod, Massaschusetts to northern Mexico and into the Caribbean. This wide range crosses several biogeographic boundaries which are coincident with genetic structuring within numerous species originating in the Pleistocene. In addition, the more northerly reaches of this species range have been further subject to the evolutionary consequences of Pleistocene glaciation due to local extinction and recolonization of formerly glaciated sites. C. variegatus thus provides an excellent vertebrate model system within which to test the extent of genetic differentiation among populations in a dominant coastal ecosystem and examine patterns of historical demography in populations distributed along a latitudinal gradient. Using mitochondrial control region and ND2 sequence data, we discovered monophyletic clades within C. variegatus with divergence times within the Pleistocene, and very low gene flow between most sites. Intraspecific genetic breaks appear to correspond broadly to biogeographic or oceanic boundaries. Pleistocene climate change appears to have had dramatic impacts on the size and distribution of populations within and near the glacial margins, but has also affected populations far from formerly glaciated regions.